Tuberculosis (TB) remains a global health burden, causing over 1.5 million deaths annually. Pathogenesis requires, among many other factors, the bacteria to grow and divide. Yet regulators of the cell cycle for Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, remain undiscovered. In my thesis work, I seek to identify the molecular mechanisms governing the cell cycle in mycobacteria. Mycobacteria grow and divide asymmetrically, in part reflecting a unique cell growth cycle characterized by windows of differential growth rates at the old and new poles of the rod shaped cell (manuscript in preparation). In mycobacteria, asymmetric growth and division is important because it rapidly generates phenotypically different cells able to withstand different stressors like antibiotics. Th model bacterium, Caulobacter crescentus, also generates phenotypically distinct daughter cells in part through asymmetric division and partitioning of key cell cycle regulators. In Caulobacter, the essential housekeeping protease complex ClpXP degrades a key cell cycle regulator to initiate this process. I postulated that similar mechanisms might underlie asymmetric growth and division in mycobacteria. ClpX, an AAA+ ATPase of the HSP100 family of molecular chaperones, can either function alone to unfold proteins or deliver unfolded substrates to ClpP for degradation. I have demonstrated that depletion of ClpX in mycobacteria results in cell cycle arrest during division using dynamic live cell imaging, supporting a role for ClpX in cell cycle regulation. Based on my phenotypic studies and the known cellular functions of ClpX, I hypothesized there is a substrate of ClpX that is a key cell cycle component. To identify ClpX substrates required for cell cycle regulation, I used a pseudo-trap in Mycobacterium smegmatis (Msm) and uncovered 200 high-confidence putative ClpX substrates. I have selected 10 putative substrates with a role in cell cycle progression to validate as substrates of ClpX. I propose ClpX regulates the mycobacterial cell cycle by acting on one or more of these identified putative substrates. I will confirm a role in cell cycle progression for the interaction of ClpX an one or more of these substrates through these aims. First, I will determine which substrates ClpXP degrades and which ClpX regulates as a chaperone. Then I will show physiologic relevance of ClpX and its substrates in cell cycle progression. These studies will provide a methodology for biochemical identification of high-confidence ATPase/chaperone substrates, and lead to a better understanding of the mycobacterial cell cycle. In completing these steps, I will gain expertise in biochemistry, molecular biology, live cell microscopy, analysis, and scientific method development.